Nanovesicles with high antioxidant and UVB protective activities made by unique biomaterials from halophilic archaebacteria

APEZTEGUIA, G; ROMERO, EL; MORILLA, MJ

Congreso; XVII Brazil MRS Meeting - SBPMat; 2018

IntroductionPremature skin aging and development of malignant cutaneous tumors, melanoma and non-melanoma, are interrelated issues that are increasingly important problems in the field of dermatology. The photoaging and photocarcinogenesis mechanism are primarily due to solar ultraviolet (UV) radiation. UV radiation alters DNA structure/cellular homeostasis and induces reactive oxygen species (ROS), which together alter signal transduction pathways and inflammatory cascade and induce immunosuppression and extracellular matrix remodelling. Current photoprotection strategies include the blockade of UV photon incidence, DNA repair, removal of ROS (antioxidant), anti-inflammation, and immunomodulation. Archaea is a group of microorganisms, including methanogens, halophiles and thermophiles that have RNA sequences, coenzymes and membrane composition different form all another organism. Archaea is an ancient form of life in earth, more related to animals and plants than to bacteria that evolved separately from bacteria and glue-green algae. Archaeal cells have unique properties separating them from the other two domains of life, Bacteria and Eukarya.Extreme halophilic archaea (salt lovers) grow in extreme conditions including salt concentrations approaching saturation, elevated temperatures and high levels of UV radiation such as natural brines, alkaline salt lakes, the Dead Sea and marine solar salterns, places non-viable for most of life. Halophilic archaea use some strategies for coping with these multiple stresses, including the production of unique group of protective molecules. For example, polar archaeolipids of membranes are composed of saturated isoprenoid chains linked via ether bonds to the glycerol carbons at the sn 2,3 position. In contrast to conventional phospholipids, polar archaeolipids are less permeable to protons, and highly resistant to hydrolytic, oxidative and enzymatic attack [1]. Neutral archaeolipids of membranes, on the other hand, are a mixture of red pigments known as carotenoids compounds that help archaea to combat the intense UV radiation [2]. Carotenoids are hydrophobic compounds and generally consisting of a C50 hydrocarbon backbone such as bacterioruberin. Our hypothesis is that nanoformulation of both polar and neutral lipids from halophilic archaea could be used as topical photoprotective agents. In this work, we have optimized the production of biomass of halophilic archaea by a simple batch bioprocess. Then we have implemented an extraction process of polar and neutral lipids using bio-solvents. Finally, we have formulated that extract into nanovesicles and test their antioxidant and photoprotective capacity on skin cells irradiated with UV light. MethodsHalophilic archaea were grown in basal medium supplemented with yeast extract and glucose. Optimal cultivation conditions (temperature, pH, NaCl concentration and C source) were screening by culturing archaea in 96 wells microtiters plates or Erlenmeyer flasks in a rotary shaker. The OD600nm was measured once a day to follow the growth. For lipid extraction, biomass was grown in 15 L medium in a 25 L home-made stainless-steel bioreactor and harvested after 96 h growth. Polar and neutral lipids were extracted from biomass using ethyl acetate and ethanol as alternative solvents to the conventional Bligh and Dyer method. Lipids, sugars and proteins in the organic and aqueous phases extracted were quantified by a colorimetric phosphate microassay, the BCA method and the phenol-sulphuric acid method, respectively. Lipids extracted were analysed by thin layer chromatography (TLC) and electrospray-ionization mass spectrometry (ESI-MS).Nanovesicles made of total lipids (polar and neutral lipids) were prepared by the thin film method. Briefly, the solvent of lipid mixtures was eliminated by N2 flush. The resulting film was resuspended with magnetic agitation in 10 mM 0.9 % w/v NaCl pH 7.4 Tris buffer up to a total lipid concentration of 10 mg/ml. Nanovesicles was characterized in terms of particle size and zeta potential. The in vitro scavenging capacity of nanovesicles against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2?-azinobis (3-ethylbenzothiazoline-6 sulfonic acid) (ABTS) radicals was measured. The photoprotective and antioxidant activities of nanovesicles on human keratinocytes cells (HaCaT) and dermal human fibroblast irradiated with UVB-UVA at DL50 (dose that cause 50% death) were measured by MTT assay and by measuring the generation of ROS using the carboxy-H2DCFDA dye, respectively.ResultsOptimization of culture parameters resulted in a productivity of 0.7 g dry biomass/L. The Bligh and Dyer method that uses methanol and chloroform has been considered as the standard method for the extraction of total lipids from microorganism. Nowadays, the use of hazardous and toxic solvents is considered as a very important problem for the health and safety of workers and environmental concerns. In this work, we compare the performances of ethyl acetate and ethanol to substitute chloroform?methanol mixtures for the extraction of halophilic archaea total lipids. Results showed that ethyl acetate-ethanol could be as effective as chloroform-methanol in terms of yield and selectivity to render a total lipid extract. The extract showed the presence of the main polar (phosphatidylglycerophosphate methylester, sulfated diglycosyldiphytanylglyceroldiether, phosphatidylglycerol and bisphosphatidylglicerol) and neutral lipids (bacterioruberin). Nanovesicles prepared with this lipid extract resulted homogeneous and negatively charged: showed a mean particle size of 280 nm with polydispersity index of 0.5 and zeta potential of -42 mV. The percentage of cell viability decreased with increasing UVB-UVA doses. Nanovesicles showed significant protection against detrimental effects of UVB-induced cell killing and ROS release. Nanovesicles were also able to quench 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals.ConclusionsThe results suggested that the halophilic archaebacteria is a sustainable source of unique biomaterials, polar and neutral archaeolipids, that can be extracted by ecological solvents and formulated in vesicles that we propose as potential new photoprotective agents.